NeXagen utilizes SELEX technology (Systematic Evolution of Ligands by EXponential enrichment) to select single-stranded oligonucleotides that have high affinity and specificity for medically relevant molecular targets. Such high-affinity oligonucleotides have commercial potential as a new class of therapeutics and diagnostics. A current bottleneck to developing the full commercial potential of the selected oligonucleotides is determining the structural features that enable them to bind with high affinity and specificity and efficient production of the selected oligonucleotides. The specific aims of the proposed project are: l) identification of structural features that give rise to the high affinity and discrimination of a RNA molecular diagnostic, 2) development of NMR as an analytical probe of conformation of potential RNA therapeutics and 3) optimization of RNA production by in vitro transcription. If successful, this work will establish the feasibility of applying NMR technology as a simple tool for rapidly screening candidate RNA therapeutics for conformational homogeneity and high stability, for elucidating structures of oligonucleotides with potential therapeutic value, and for assessing the critical structural features involved in their binding to their target molecules. It will also demonstrate whether structural features that bring about the high binding and specificity of oligonucleotides for their targets can be identified by NMR spectroscopy. Further, it will demonstrate the feasibility of proposed approaches for production of RNA by in vitro transcription. In Phase I, these methods will be applied to a model system: theophylline:RNA ligand. If they are shown to be feasible, Phase II will involve application of the described methods to commercializing oligonucleotides of medical and commercial value in combating AIDS, cardiovascular disease, and tumor metastasis.